Review on Photomicrography based Full Blood Count (FBC) Testing and Recent Advancements

With advancements in related sub-fields, research on photomicrography in life science is emerging and this is a review on its application towards human full blood count testing which is a primary test in medical practices. For a prolonged period of time, analysis of blood samples is the basis for bio medical observations of living creatures. Cell size, shape, constituents, count, ratios are few of the features identified using DIP based analysis and these features provide an overview of the state of human body which is important in identifying present medical conditions and indicating possible future complications. In addition, functionality of the immune system is observed using results of blood tests. In FBC tests, identification of different blood cell types and counting the number of cells of each type is required to obtain results. Literature discuss various techniques and methods and this article presents an insightful review on human blood cell morphology, photomicrography, digital image processing of photomicrographs, feature extraction and classification, and recent advances. Integration of emerging technologies such as microfluidics, micro-electromechanical systems, and artificial intelligence based image processing algorithms and classifiers with cell sensing have enabled exploration of novel research directions in blood testing applications.

Cell Detection in Knife-Edge Scanning Microscopy Images of Nissl-stained Mouse and Rat Brain Samples Using Random Forests

Microscopy has developed into a very powerful medium for studying the brain. The Knife-Edge Scanning Microscope (KESM), for example, is capable of imaging whole rat and mouse brains in three dimensions, and produces over 1.5 terabytes of images per brain. These data can reveal the structure and organization of the brain's internals including neurons and blood vessels. Neuron count and density strongly influence the behavior of an organism, and measuring their spatial distribution is key to a better understanding of the workings of the brain. This kind of analysis involves identifying neurons in large brain regions, for which fast automated detection methods are necessary. Most of the current automated cell detection techniques require complex preprocessing of images, use heuristics that are time consuming to develop, or do not generalize well to three dimensional data. In this thesis, I propose two methods based on random forests for detecting neuron bodies in the rat and mouse brain KESM data. The proposed methods require a few hundred cell centers to be manually labeled. Random forests are trained to predict if a voxel is a cell center or not by using these labeled data and features derived from orthogonal image patches. They can then be used to locate cell centers in 3-D in other images, aided by a refinement step whose parameters are determined from the training data. Minimal manual input is required, and random forests provide a good combination of accuracy and speed. This is expected to enable fast counting and density measurements of neurons in brain regions. The detected cell centers should also be valuable as seeds for cell segmentation methods

Spectral Ultrasound Characterization of Tissues and Tissue Engineered Constructs.

Even though ultrasound imaging is widely used in clinical diagnosis and image-guided interventions, the field is far behind other areas of clinical quantitative image analysis, such as MRI, CT and X-ray mammography. In this thesis, non-destructive and non-invasive ultrasound characterization techniques were developed to study the tissue micro-structural details using high frequency spectral ultrasound imaging (SUSI). The techniques were explored in in-vitro conditions of acellular and cellular tissue engineered constructs and then on ex-vivo tissues for their characterization. SUSI was used to assess the amount of hydroxyl-apatite (HA) mineral, differentiate HA mineral types and study their distribution in acellular tissue engineered constructs. The process of mineral deposition from surrounding mineralizing media onto simple collagen constructs was also studied and characterized with SUSI. 3D morphological changes of the constructs with MC3t3 cells was monitored and characterized for the developmental changes such as net cell proliferation/apoptosis and cell differentiation process through mineral production by the early osteoblastic MC3t3-cell constructs in-situ. A novel method was introduced using SUSI to estimate the amount of mineral secreted by the differentiated osteoblast cells in a non-destructive method. Then, SUSI was investigated in ex-vivo cardiac tissues to monitor and characterize the cellular changes during high-intensity focused ultrasound ablation with high-frame-rate and high-resolution ultrasound imaging. The mechanistic hypotheses behind the improvement in lesion detection were investigated and best identification methods to assess lesion formation and transient gas body activities were proposed to provide a method for visualizing spatiotemporal evolution of lesion and gas–body activity and for predicting macroscopic cavity formation upon its implementation as a real-time monitoring technique with feedback control system for HIFU treatment of atrial fibrillation to improve the ablation process. Even though the results from the developed techniques show great promise in in-vitro and ex-vivo settings, additional work needs to be carried out to demonstrate the applicability of the techniques in in-vivo.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99788/1/msreddy_1.pd

A Pipeline for Automated Assessment of Cell Location in 3D Mouse Brain Image Sets

Mapping the neuronal connectivity of the mouse brain has long been hampered by the laborious and time-consuming process of slicing, staining and imaging the brain tissue. Recent developments in automated 3D fluorescence microscopy, such as serial two- photon tomography (STP) and light sheet fluorescence microscopy, now allow for automated rapid 3D imaging of a complete mouse brain at cellular resolution. In combination with transsynaptic viral tracers, this paves the way for high-throughput brain mapping studies, which could greatly advance our understanding of the function of the brain. Because transsynaptic tracers label synaptically connected cells, the analysis of these whole-brain scans requires detection of fluorescently labelled cells and anatomical segmentation of the data, which are very labour- and time-intensive manual tasks and prevent high-throughput analysis. This thesis presents and validates two software tools to automate anatomical segmentation and cell detection in serial two photon (STP) scans of the mouse brain. Automated mouse atlas propagation (aMAP) segments the scans into anatomical regions by matching a 3D reference atlas to the data using affine and free-form image registration. The fast automated cell counting tool (FACCT) then detects fluorescently labelled cells in the dataset with a novel approach of stepwise data reduction combined with object detection using artificial neuronal networks. The tools are optimised for large datasets and are capable of processing a 2.5TB STP scan in under two days. The performance of aMAP and FACCT is evaluated on STP scans from retrograde connectivity tracing experiments using rabies virus injections in the primary visual corte

Diels Alder-mediated release of gemcitabine from nanoparticles: developing improved methods for pancreatic cancer drug delivery

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC), is the deadliest type of cancerous malignancy with a survival rate of only 3.7 % after 5 years. The first line current treatment is a drug called gemcitabine, which has been shown to display effectiveness in only 23.8 % of patients. Hybrid nanoparticles (HNPs) comprised of an iron oxide core and outer gold coat have shown great potential for anti-cancer therapies. The magnetic iron oxide cores and the surface plasmon resonance (SPR) properties of the gold surface provide the HNPs with the capabilities of diagnostic imaging and drug delivery, making them true theranostic agents.A novel thiolated thermally labile drug (TTLD) analogue of gemcitabine was successfully synthesized and attached to the surface of HNPs forming a novel drug formulation called TTLD+HNP. This TTLD compound is comprised of a gemcitabine molecule with a Diels Alder cycloadduct. Gem-Mal, a maleimide derivative of gemcitabine is released during retro Diels Alder (rDA). The mode of release involves heat-activation of the rDA reaction facilitated by the SPR of the gold shell. TTLD was characterised with mass spectrometry, nuclear magnetic spectroscopy and IR spectroscopy.Preliminary studies determined that the TTLD compound doesn’t undergo rDA at 20 °C and a subsequent 4 week study displayed no rDA occurring at 20 °C. Afterwards in vitro experiments including the MTT and trypan blue assay determined that Gem-Mal is 4.6 times less cytotoxic than gemcitabine but is taken up by cells 11 fold faster when attached to the HNPs. Upon heat-activation at 44 °C, the TTLD+HNP formulations cytotoxicity increased by 56 % outperforming gemcitabine by 26 %, confirming its temperature driven activity. The TTLD+HNP drug formulation is the first of its kind and has displayed superior anti-cancer activity to the current first line drug gemcitabine after heat mediated controlled release

Dual Phase Engineered Tissue for Enhanced Bone Formation.

Large bone defects are a significant clinical problem in the United States and worldwide. “Non-unions” are fractures that fail to heal due to a lack of blood supply to the defect site. In our approach to bone regeneration, we create modular engineered tissues (“microbeads”) designed to form bone, and combine them with a surrounding vascularizing tissue to generate a dual-phase injectable matrix for enhanced bone formation. In the first Aim, human bone marrow mesenchymal stem cells (bmMSC) or human adipose stem cells (AdSC) were embedded in collagen/fibrin (COL/FIB) or collagen/fibrin/hydroxyapatite (COL/FIB/HA) microbeads. Both cell types mineralized microbeads, indicating differentiation towards the osteogenic lineage. The second Aim used a co-culture model of bmMSC and human umbilical vein endothelial cells in COL/FIB composite hydrogels to create a vasculogenic matrix. Cell ratio and matrix composition were varied in a systematic manner. Vascular network formation increased in vitro with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited similar responses. Hydroxyapatite (HA) was found to recover endothelial network formation in unconstrained hydrogels. Over 7 days of dorsal subcutaneous implantation in nude mice, these matrices exhibited increasing neovascularization, though there was no significant effect of HA. The final Aim combined osteogenic microbeads with a surrounding vasculogenic matrix to evaluate the effect of this dual-phase tissue in vivo. Both vasculogenesis and osteogenesis were examined in a subcutaneous bone formation model in the mouse at 4 and 8 weeks. Blood flow measured by Doppler imaging was not significantly different between any conditions at any time point, except at 8 weeks where the vasculogenic matrix alone was lower than all other groups. Micro-computed tomography of ectopic bone demonstrated significantly higher bone volume in the osteogenic microbead condition at 4 weeks and both the blank and osteogenic microbead conditions at 8 weeks, compared to the dual osteogenic/vasculogenic condition. These data suggest an inhibitory effect of the vasculogenic component on bone formation in the non-ischemic model. Dual-phase implants may be more effective in ischemic orthotopic bone regeneration models, and these results demonstrate that such constructs can be designed, fabricated, and delivered for therapeutic use.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99786/1/ramrao_1.pd